Dried blood factor composition trehalose

ABSTRACT

A stable blood factor composition contains a stabilising amount of trehalose in the absence of human serum albumin to provide a product stable at up to 60° C.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending applicationU.S. Ser. No. 09/888,734, filed Jun. 25, 2001; which is a continuationapplication of U.S. Ser. No. 08/875,796, filed Oct. 30, 1998, now U.S.Pat. No. 6,649,386, which was filed as a National Stage Application ofInternational Application Number PCT/GB96/00119, filed Jan. 19, 1996,published, pursuant to PCT Article 21(2); which claims priority to GreatBritain Application GB 9501040.1, Jan. 19, 1995.

BACKGROUND OF THE INVENTION

This invention relates to dried compositions of blood factors forreconstitution with water or aqueous solutions.

Blood factors, particularly factor VIII and factor IX, are now thestandard treatment for diseases caused by a lack of the appropriatefactor, in particular haemophilia. The blood factor has generally beenderived from human blood by various extraction techniques, for exampleas disclosed in EP-A-0083483, or by expression in genetically modifiedmicroorganisms, for example as disclosed in EP-A-0160457 andEP-A-0182448.

Blood factor products such as factor VIII are highly delicate, unstableproteins. They are usually supplied in the form of frozen solutions inan appropriate buffer or, more generally, as freeze-dried powders. Eventhe freeze-dried powders must be kept cold during storage. In order tostabilise the freeze-dried material, commercial products contain astabilising protein, in particular human serum albumin (HSA). It has notbeen thought possible to prepare a dry blood factor composition which isstable at ambient temperatures and at pasteurisation temperatures (e.g.60° C.) in the absence of HSA. However, the presence of HSA introducesconsiderable problems of purification since it is essential that theprotein is free of viral contamination. The use of recombinant HSA toovercome these problems is expensive.

Trehalose is known to be a highly effective stabilising agent fordelicate proteins, as disclosed in U.S. Pat. No. 4,891,319, enablingproteins to be dried at temperatures above freezing. We have now foundthat if trehalose is used to stabilise a blood factor product, not onlycan the product be dried with or without freezing, but also the productis stable even when retained at a temperature of 60° C. for an extendedperiod, in the complete absence of HSA. According to the presentinvention therefore we provide a stable dried blood factor compositioncontaining a stabilising amount of trehalose in the absence of albumin.

In general, any stabilising amount of trehalose may be used and anexcess in general causes no problems. Indeed, the presence of trehaloseaids the rehydration process and is physiologically acceptable forinjection, being rapidly metabolised to glucose. The composition isparticularly suited to formulations of factor VIII, which may alsocontain appropriate buffering and ion-reinforcing salts, in particular asource of calcium. In general, a ratio of about 1.0 to 1.5 mg of calciumions per unit of factor VIII is appropriate.

Other buffering and modifying agents may also be present in the driedmaterial for reconstitution to the injection solution, for examplehistidine. However, we have found that the level of salts, particularlysodium chloride, present can affect the preservation on drying. It isthought desirable for the commercial product for injection to have anisotonic salt concentration. However, the processing formulations whichare freeze-dried are desirably hypertonic, typically containing about500 mM NaCl (isotonic NaCl=150 mM), as this is considered to helpstabilise the blood factor. As a result, commercial freeze-driedformulations generally have a high salt content and are reconstitutedfor injection with the appropriate amount of sterile water to obtain anisotonic solution.

A considerably reduced salt content is preferred for the dried materialof the invention and, in general a solution of about 500 units of FactorVIII per ml to be dried should preferably contain less than 200 mM e.g.75 to 150 mM, NaCl, especially about 100 mM, or even lower, e.g. 20 to50 mM, especially about 22 to 30 mM. Low salt preparations possess ahigher dry stability. The dried product can be reconstituted to thedesired salt level with a saline solution instead of the conventionalwater. In general, the molar ratio of trehalose to salt should be above1:1, especially above 2.5:1 e.g. above 10:1, preferably above 12.5:1.

The dried composition may be obtained by drying an appropriate solutionof the blood factor containing the correct proportions of trehalose andother desired components. In general, the solution that is dried shouldsimply contain all the components required in the reconstitutedinjection solution, although the solution for drying may not necessarilybe at the same dilution. Typically, the solution for drying will containfrom 1 to 1000 units of factor VIII per ml. The methods of drying mayinclude freeze drying, vacuum drying and spray-drying. A particularlypreferred method according to the invention comprises vacuum drying at atemperature no greater than 25° C., preferably no greater than 10° C.,to form a foam, thus maximising the exposed surface and the dryingeffect.

The following examples illustrate the invention further.

EXAMPLE 1

Recombinant factor VIII was received as a deep frozen solutioncontaining approximately 2000 to 2500 units/ml in the manufacturer'shigh salt buffer. The thawed solution was dialysed against a buffersolution containing 500 mM NaCl, 15 mM CaCl₂ and 10 mM histidine at pH6.8. The dialysed protein was diluted in the same buffer, but with addedtrehalose to give a final concentration of 500 units per ml and 10% byweight trehalose at pH 6.8. This solution was vacuum dried in 1 mlaliquots. Vacuum was reduced step-wise from atmospheric to 4 Pa (30mTorr) to avoid freezing the sample. The temperature of the sample wasnot allowed to rise above 12° C. until the formation of a foam, afterwhich the temperature was kept below 30° C. Total drying times were 24to 28 hours.

The samples were stored for 0, 1.5, 3 and 6 months at 40° C. and thenreconstituted in 5 ml aliquots of sterile distilled water before beingtested for activity. The results are shown in the following table incomparison with a commercial freeze-dried product containing HSA. Boththe test and commercial samples have a high salt content. Thepost-drying results show that with trehalose it is possible to dryfactor VIII successfully in the absence of HSA, but that a high saltcontent is unsatisfactory for long term storage, even in the presence ofHSA.

Percentage of initial activity Time (months) Sample Commercial Product 0100.0 100.0 1.5 86.8 95.3 3 75.1 71.2 6 76.6 63.6

EXAMPLE 2

Samples were dried as described in Example 1 but using a bufferformulation comprising 100 mM NaCl, 15 mM CaCl₂, 15 mM histidine and1.27 molar trehalose (48%) and stored at 60° C. before reconstitution.The results are given in the following table, in which the activity ismeasured on an ACL 100 automated coagulometer (InstrumentationLaboratory SpA, Milan, Italy). The test sample, with a low salt contentshowed no significant loss of activity on storage, even after four weeksof 60° C.

Percentage of initial activity recovered Wet control 100.0 Post-drying95.5 Two weeks 96.0 Four weeks 96.8

EXAMPLE 3

Two formulations were prepared containing different salt concentrationsas shown:

Formulation A Formulation B NaCl  0.13% NaCl  1.03% CaCl₂ 0.011% CaCl₂0.011% L-histidine  0.12% L-histidine  0.12% Tris 0.002% Tris 0.002%Tween 80 0.002% Tween 80 0.002% PEG 3350 0.004% PEG 3350 0.004%Trehalose  7.5% Trehalose  7.5% Factor VIII 50 U/ml Factor VIII 50 U/mlWater to 100% Water to 100%

10 ml portions of the formulations were dispensed into separate 30 mlvials so as to give a concentration of factor VIII of 500 units/vial.

Freeze-drying was performed in a Laconco (Lyph-lock 12 stoppering)freeze drier. Initially, the samples were cooled to −40° C. and thenplaced under vacuum, before being warmed to −35° C. After 80 hours, thesamples were warmed at a rate of 2.5° C./h until the shelf temperaturereached 25° C. The samples were then kept at 25° C. for two hours beforebeing sealed under vacuum and removed from the drier. After drying, thesamples were rehydrated with 10 ml of water and the concentration offactor VIII was measured twice (Assays 1 and 2). The results are givenin the following table and are expressed as a percentage of theconcentration of Factor VIII with respect to the prefill control (whichhad been frozen at −70° C.). From the results shown, it can be concludedthat Factor VIII can be successfully freeze dried in a trehalose basedformulation in the absence of HSA.

Sample Assay 1 Assay 2 Formulation A 77% 85% Formulation B 91% 94%

1. A vial containing a dried native Factor VIII composition comprising aFactor VIII-stabilizing amount of trehalose and no albumin, thecomposition being (a) stable without refrigeration, and (b) suitable forreconstitution with water or with an aqueous solution for administrationto a hemophilic patient by injection.
 2. The composition of claim 1,containing 0.15 to 2.5 mg trehalose per unit of Factor VIII.
 3. Thecomposition of claim 1, wherein said native Factor VIII is recombinant.4. The composition of claim 1, containing 1.0 to 1.5 mg Ca²⁺ per unit ofFactor VIII.
 5. The composition of claim 1, wherein the compositioncomprises salt and the molar ratio of trehalose to the salt is above1:1.
 6. The composition of claim 5, containing more than 2.5 molestrehalose per mole of salt.
 7. The composition of claim 6, containingmore than 10 moles trehalose per mole of salt.
 8. A process forpreparing the dried native Factor VIII composition defined in claim 1which process comprises drying a solution of native Factor VIII andtrehalose in the absence of albumin.
 9. The process of claim 8, whereinthe drying is done at a temperature of no greater than 10° C.
 10. Theprocess of claim 9, wherein the drying is freeze-drying.
 11. The processof claim 8, wherein an aliquot of the solution is placed in a vial andis freeze-dried.
 12. A method for preparing a Factor VIII therapeuticcomposition for administration to a patient wherein the method comprisesreconstituting the composition defined in claim 1 to obtain a solutionthat is suitable for injection.
 13. The method of claim 12, wherein thereconstituting with water.
 14. The method of claim 12, wherein thereconstituting is with saline.